This proposal will develop a novel cancer immunotherapy that uses scFv-NKG2D to treat liquid and solid tumors. The scFv-NKG2D is a Bispecific T cell Engager (also referred to as a BiTE) that uses an anti-CD3 scFv linked to the extracellular portion (EC) of NKG2D to engage T cells with NKG2D ligand+ tumor cells. BiTE molecules with this design do not activate T cells until they bind to ligand+ tumor cells. This molecule lacks an Fc portion so it cannot interact with Fc receptors, which reduces off-target effects. NKG2D ligands are expressed on many types of tumor cells but rarely normal cells, which provide a relatively specific way to target many kinds of tumor cells. In addition, data indicate that T regulatory (Treg) cells and myeloid-derived suppressor cells (MDSCs) can express NKG2D ligands within the tumor microenvironment, so that by targeting NKG2D ligands it may also lead to elimination of these immune suppressive cells and promote anti-tumor immunity. We have developed both various murine and human versions of scFv-NKG2D that bind to murine or human CD3 and murine or human NKG2D ligands. Because of the species specificity of these molecules, we can use these other versions as control proteins. We will determine efficacy of scFv-NKG2D with primary human T cells and autologous tumor cells, and we will determine ways to enhance efficacy by increasing T cell function and trafficking. This proposal has three specific aims: Aim 1: To determine the efficacy of scFv-NKG2D therapy against solid and hematological tumors. Aim 2: To determine efficacy against human tumors and off-target effects of scFv-NKG2D therapy. Aim 3: To enhance the efficacy of scFv-NKG2D therapy by removal of immunosuppressive mechanisms. We have obtained preliminary evidence for efficacy of scFv-NKG2D against solid and liquid types of tumors, and data indicate that merely 5g i.v. significantly enhances survival. We have personnel that have extensive experience working with NKG2D, murine tumor models, and studying immune mechanisms. This immunotherapy approach has the potential to be beneficial against a wide variety of tumors because of the broad expression of NKG2D ligands on many tumors. Because it also targets immunosuppressive cells within the tumor microenvironment, scFv-NKG2D treatment may prove more efficacious than approaches that only target the tumor cells themselves. We have an expert team of clinical and scientific collaborators involved in these studies, so we will be ina strong position to create a development plan for phase I clinical testing.